future science group 523ISSN 1759-726910.4155/BFS.13.38 © 2013 Future Science Ltd

Improving lignocellulolytic enzyme production with Penicillium: from strain screening to systems biology

Guodong Liu1, Yuqi Qin1,2, Zhonghai Li1 & Yinbo Qu*1,2

Many Penicillium species produce enzyme systems with good performances in lignocellulose degradation. In our laboratory, lignocellulolytic enzyme-producing Penicillium oxalicum (formerly classified as Penicillium decumbens) strains have been studied for more than 30 years. High cellulase-producing mutants have been obtained through random mutagenesis and genetic engineering, and the components in the enzyme systems have been elucidated using systems biology tools. The effects of different carbon sources on the production level of lignocellulolytic enzymes have been studied, and the related molecular mechanisms have been investigated. When compared with the widely used cellulase producer Trichoderma reesei, some unique features have been found in P. oxalicum, including higher b-glucosidase activity, higher numbers of lignocellulolytic enzyme gene, and different response of cellulase gene expression to some disaccharides. To boost the economic potential of the biorefineries using lignocellulosic biomass, P. oxalicum strains need to be further improved regarding the performance and production level of the enzyme systems.

Review

1State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, PR China 2National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, PR China *Author for correspondence: Tel.: +86 531 88365954; Fax: +86 531 88565610; E-mail: quyinbo@sdu.edu.cn

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One of the key technologies in biofuel production is the degradation of complex lignocellulosic bio­mass to monosaccharides. Lignocellulolytic enzyme systems, mainly produced by filamentous fungi in industry, are widely used in this process. To achieve efficient hydrolysis of lignocellulosics, high dosages of lignocellulolytic enzymes are needed due to the natural recalcitrance of the materials [1,2]. Thus, the high cost of lignocellulolytic enzymes is now a major barrier in the economically competitive production of biofuels from lignocellulosic materials [3]. Trichoderma reesei is now the main source of commercial lignocellulolytic enzymes [4]. Accordingly, T. reesei has become a paradigm for researches on lignocellulolytic enzymes and the regulatory mechanisms controlling their synthesis in the scientific community [5].

Compared with that of T. reesei, lignocellulolytic enzyme systems produced by many Penicillium species have generally better performances (higher cellulose conversion at equal protein loadings) in lignocellulose

hydrolysis [6–9]. Some Penicillium species (e.g., Penicillium funiculosum [10]) have been used for the production of commercial lignocellulolytic enzymes. The related reports about the production levels, hydrolysis efficiencies and industrial traits of crude lignocellulolytic enzyme systems from Penicillium strains have been reviewed [6]; however, knowledge on the composition of these enzyme systems and the regulation of their production is still poor compared with those in T. reesei and Aspergillus species, which significantly limits the progress of further strain improvement.

In our laboratory, a lignocellulolytic enzyme­producing Penicillium has been studied for more than 30 years. The fungus was first identified to be P. decumbens, and was recently reclassified to be P. oxalicum based on sequence ana lysis (Figure  1) [11,12]. Several carbon catabolite repression (CCR)­resistant mutants with higher cellulase productivities were obtained, and have been used for industrial­scale cellulase production and pilot­scale cellulosic ethanol production in China.

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The whole­genome sequence of P. oxalicum was determined in 2009, which allowed the application of systems biology tools on it. In this review, we summarize the progress of our research on P. oxalicum strains. The studies include the development of lignocellulolytic enzyme over­producing mutants, biochemical characterization of enzyme components, exploration of the cellular network controlling enzyme synthesis and application of the enzymes in biorefinery. Results based on systems biology studies will be highlighted.

Screening & development of P. oxalicum strains for cellulase productionThe wild­type P. oxalicum isolate 114 was screened from decayed straw­covered soil in the suburb of Jining, east China, in 1979 using double­layer agar plates [13]. Ball­milled holocellulose, instead of pure cellulose, was used as the carbon source in the screening plates, ensuring the screened isolates (forming hydrolytic halos) could produce complete cellulolytic and hemicellulolytic enzyme systems. Several single­spore isolates with similar cellulase production levels were purified from 114. One of the purified strains, 114–2, was kept in our laboratory as the wild­type strain and used in most subsequent studies (Figure 2). When 114–2 was cultivated in cellulose­containing liquid medium, cellulase, xylanase, amylase and pectinase activities were detected.

Production of cellulase and hemicellulase by the wild­type P. oxalicum strain was severely repressed in the presence of excessive glucose or glycerol. After multiple rounds of mutagenesis of isolate 114 using ultraviolet irradiation and nitrosoguanidine, a mutant JU1 with the ability to produce cellulase in glucose­containing medium was obtained (Figure  2) [13]. In

glucose­free medium (containing 2% holocellulose plus 0.5% wheat bran), JU1 also produced higher cellulase activity (3.1 units of filter paper activity [FPA] per ml) than 114–2 did (0.8 U ml­1). Notably, JU1 showed quite different morphology from 114–2, including slower hyphal extension, thicker hyphae, loss of bluish­green conidial pigment, lower amounts of conidia production and circumscribed pink colonies.

JU1 was then adapted repeatedly on spent ammonium sulfite liquor (SASL; one kind of pulp mill effluent) gradient agar plates to improve its tolerance to this toxic liquor [14]. The experimental evolution generated a SASL­tolerant mutant JU­A10. JU­A10 grew faster than JU1 in SASL, synthetic high­sulfate medium and even nontoxic glucose medium [15]. The time of maximum cellulase production by JU­A10 was 20 h earlier than that of JU1 in SASL­waste fiber medium [14]. Through genome shuffling, three fusants with more than twofold higher cellulase (FPA) productivities were obtained from JU­A10 [16]. In addition, several other cellulase high­producing mutants, such as JU­A10–1 [17], JU­140–12 [18] and JU­A10­T [19], were also derived from JU­A10 by further mutagenesis and screening (Figure 2).

Rational strain engineering of P. oxalicum was performed based on the clear genetic background provided by genome sequencing. The assembled genome size of wild­type strain 114–2 was 30.19 Mb, which was predicted to encode 10,021 proteins [20]. For mutant JU­A10­T, the genome size was 30.69 Mb, with 10,473 protein­coding genes predicted [19]. To our knowledge, these are the first genome sequences of industrial cellulase­producing Penicillium species. Homologs of most proteins known to affect cellulase production in fungi were annotated in P. oxalicum, some of which were targeted for the purpose of strain improvement. The gene encoding the ortholog of Neurospora crassa NCU05137 (a secreted protein whose deletion led to increased cellulase expression [21]) in P. oxalicum, PDE_01641, was deleted in both 114–2 and JU­A10­T [22]. Deletion of the PDE_01641 gene increased cellulase (FPA) production and cell growth on cellulose in 114–2, and resulted in a 36% increase in cellulase production at 48 h of fermentation in JU­A10­T. Deletion of gene bgl2 (encoding major intracellular b­glucosidase) improved the final cellulase activity (after 144 h of fermentation in the medium containing 1% cellulose plus 1% wheat bran) to 0.88 U ml­1, which was 3.3­fold that of 114–2 [23]. Combined manipulations of some crucial (positive or negative) transcription regulators resulted in an up to tenfold elevation of cellulase production in 114–2 [Li Z, Qu Y,

Unpublished Data]. The engineering strategies are being implemented in industrial hyper­producing mutants to further improve production levels.

Key terms

Lignocellulolytic enzyme system: Mixture of many kinds of enzymes acting together for the degradation of complex lignocellulosics. Cellulases, hemicellulases and ligninases are the main components.

Penicillium: Genus of fungi belonging to the division Ascomycota, order Eurotiales, class Eurotiomycetes, family Trichocomaceae. Penicillium species are widely distributed in the natural environment, and have been used in the production of drugs, food and enzymes in industry.

Carbon catabolite repression: A regulatory system ensuring that micro organisms use rapidly metabolized carbon sources (e.g., glucose) first. When these carbon sources are present, the transcription of genes encoding enzymes for the utilization of alternative carbon sources (e.g., cellulose) are repressed.

Systems biology: Study aimed at under standing how all the components in biological systems (cell, tissue, organism, and so on) function together. Genome-wide experimental techniques are widely used for systems biology studies.

Holocellulose: Total polysaccharides (cellulose and hemicellulose) of wood or other lignocellulosic materials. Holo cellulose can be prepared from natural lignocellulosic materials by removing extractives and lignin using chemical reagents (e.g., acidified sodium chlorite).

Filter paper activity: Widely used, standard representation of cellulase activity. Filter paper (a blend of amorphous and crystalline cellulose) is hydrolyzed and the amount of reducing sugars released is determined for activity calculation.

Genome shuffling: Technique recom-bining multi-parental genome sequences to concentrate beneficial mutations in one genome for strain improvement.

Homolog: Genes or proteins of shared ancestry. Homologs that originated by vertical descent from a common ancestor are called orthologs.

Nonhomologous endjoining pathway: A cellular process ligating dsDNA ends without the need for sequence homology.

Processivity: For cellulases, processivity is the ability of a cellulase to continuously hydrolyze one cellulose chain without dissociating from it. Cellobiohydrolase I from Trichoderma reesei is a typical processive cellulase.

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Strains with specific genotypes were also developed to facilitate the engineering and functional genomics study in P. oxalicum. A strain Dpku70, in which the nonhomologous endjoining pathway was disrupted, was constructed to improve gene­targeting efficiency [24]. The frequency of homologous recombination in Dpku70 reached 100%, much higher than that in the parent wild­type 114–2 (20–90%, depending on genes). A pyrG­auxotrophic mutant M12 was isolated through the spontaneous mutagenesis of 114–2 [25]. The pyrG marker can be selected bidirectionally (either presence or absence), thus allowing marker recycling for multiple gene manipulations [26].

Characterization of the lignocellulolytic enzyme system of P. oxalicumSome enzymatic properties of P. oxalicum enzyme systems were studied using crude enzymes. The enzyme system of mutant JU1 had optimum temperatures of 40, 55, 60 and 70°C, respectively, when absorbent cotton (hydrolyzed for 24 h), filter paper (hydrolyzed for 1 h), sodium carboxymethyl cellulose (hydrolyzed for 15 min) and salicin (hydrolyzed for 30 min) were used as the substrates for hydrolase activity assays [27]. The optimum pH of the enzyme system on filter paper was 4.8. These results are similar to those of enzyme systems from many other mesophilic fungi [28–30]. For the effect of metal ions, cellulase activity of the enzyme system of mutant JU­A10­1 was found to be inhibited by Fe3+ [31]. To get a better understanding of the lignocellulolytic enzyme

system of P. oxalicum, the composition of the enzyme mixtures and properties of single enzymes were studied.

� Studies of single-enzyme components prior to genome sequencingCellobiohydrolase (CBH; exo­acting cellulase), endo­1,4­b­glucanase (endoglucanase [EG]) and b­glucosidase (BGL; cellobiase) are the three major types of enzymes in cellulose hydrolysis [32]. When the enzyme systems of P. oxalicum were separated by polyacrylamide gel electrophoresis and then stained for enzyme activities, one to two bands for CBH activities and one band for BGL activity were detected, while more than ten bands were detected for EG activities [33]. Through a combination of several different column chromatographic procedures, six CBHs, eight EGs and one BGL were purified from strain JU1 [34]. The purified enzymes differed in molecular weight, isoelectric point, carbohydrate content, substrate specificity, specific activity, kinetic parameters, product profile and randomness (processivity) of action. The major cellobiohydrolase, CBHI (later renamed Cel7A­2), was purified from strain JU­A10 and studied for the relationship between glycosylation type and enzymatic properties [35] (for details, see the subsection ‘Post­translational modification of cellulases’). Another cellobiohydrolase purified from JU­A10, PdCel6A, was very stable at acidic pH, and thus could efficiently improve the production of ethanol from lignocellulosics under acidic conditions [36]. Using degenerate primers, one CBH gene cbh1 (later renamed cel7A-1) [37] and

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P. oxalicum 114-2 (KF152942)

P. oxalicum JU-A10-T (KB908899)

P. oxalicum NRRL 790 (AF034455)

P. oxalicum CCF:2052 (HE651146)

P. oxalicum CCF:2315 (HE651152)

P. oxalicum NRRL 787 (AF033438) TYPE

P. decumbens NRRL 741 (AF033453) TYPE

P. chrysogenum NRRL 807 (AF033465) TYPE

Figure 1. Maximum-likelihood phylogenetic tree of Penicillium strains. DNA sequences including internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2 and partial 28S ribosomal RNA gene were aligned using ClustalX 2.0 [11], and then the phylogenetic tree was generated by MEGA 5.10 [12] using the bootstrap maximum likelihood method with 1000 replicates. GenBank accession numbers are shown in parentheses. Partial nucleotide sequences of the assemblies (2493–3639 for 114–2 and 1064521–1065666 for JU-A10-T) were used for alignment. The type strains of Penicillium oxalicum, Penicillium decumbens and Penicillium chrysogenum are marked in the figure. Nucleotide sequence identities between the sequence from strain 114–2 and those of type strains are shown (numbers on the right sides of dotted lines). The identification of 114–2 and JU-A10-T as P. oxalicum was also supported by aligning the coding sequences of calmodulin, b-tubulin and RNA polymerase II subunit 2 genes with those from identified P. oxalicum strains, respectively (nucleotide sequence identities are 98–100%).

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three EG genes cel7B, cel5A and cel45A [38,39] were cloned from P. oxalicum. The three EGs were heterologously expressed and characterized in yeasts. The high hydrolytic activity of recombinant Cel7B [39] and the thermophilic property of recombinant Cel45A [38] were detected.

BGL hydrolyzing cellobiose to glucose is important for the release of product inhibition in cellulose hydrolysis [40]. Like many other Penicillium species [6], P. oxalicum produces enzyme systems with higher BGL activity than that of T. reesei, which thereby have better performances in simultaneous saccharification and fermentation of acid­pretreated corncobs to ethanol [41]. pBGL1 is the primary BGL in the enzyme system of P. oxalicum [42]. Either supplementation of purified pBGL1 to the enzyme mixture [42]

or integration of pbgl1 gene into the genome [43] could significantly enhance the saccharifying ability of the enzyme system from T. reesei.

� Global understanding using genomic & proteomic approachesIntegrated genomic and proteomic ana lysis provides a rapid and comprehensive understanding on the composition of the enzyme system of P. oxalicum [20]. Eighteen genes encoding cellulolytic enzymes, including three CBHs, 11 EGs and four polysaccharide mono­oxygenases (PMOs; previously known as glycoside hydrolase [GH] family 61 EGs [44]) were predicted in P. oxalicum (Figure 3) [45]. Some of the enzymes were subsequently heterologously expressed in yeast and characterized for their properties. One GH family 5 EG (Cel5C) containing an unusual CBM_X2 domain showed activities on both cellulose and xyloglucan [46]. Three of the four PMOs showed synergism with commercial cellulase from T. reesei in cellulose hydrolysis [Qu Y, Unpublished Data]. For other degrading enzymes, 11 BGLs, 51 hemicellulases, 25 pectinases and 11 amylolytic enzymes were predicted [20]. The higher number of hemicellulases and cellulose binding domain (CBM1)­containing proteins in P. oxalicum was highlighted in the comparison with those in T. reesei [20]. Particularly, the number of CBM1­containing proteins (n = 23) is the highest among all the so­far sequenced Aspergillus and Penicillium species (three to 22 in the remaining species).

The actual compositions of enzyme systems were further elucidated by proteomic analyses of the secretomes. Cellulases, hemicellulases, amylases and proteases were found to be the major components [19,20]. When compared with those of T. reesei [47,48], some unique features of the composition of P. oxalicum enzyme systems are noted:

� Two GH family 7 CBHs, one with and the other without CBM1 (Figure 3), are present in the secretome of P. oxalicum [19]. The later type of CBH is genetically absent in T. reesei [49];

� In addition to Cel7B (ortholog of T. reesei Cel7B/EG I), Cel5B, but not Cel5A (ortholog of T. reesei Cel5A/EG II), is another major EG in P. oxalicum [19]. It was noted that a close homolog of Cel5B was also reported to be a major EG in Penicillium brasilianum [50], but its ortholog is absent in T. reesei [20];

� A PMO (Cel61A) accounts for approximately 6% of the total secretome of 114–2 and approximately 14% of that of JU­A10­T [19], much higher than the proportions of PMOs in enzyme preparations produced by T. reesei (<1%) [47,48]. The role of this PMO in lignocellulose hydrolysis by P. oxalicum is being studied in our group;

� P. oxalicum and T. reesei each have some unique hemicellulases in their secretomes, such as feruloyl esterase and endo­b­1,4­galactanase in P. oxalicum [20], and GH74 family xyloglucanase and b­xylosidase in T. reesei [47]. The information provided a rational basis for further improvement of the enzyme systems.

� Post-translational modification of cellulasesPost-translational modifications such as glycosylation are widely detected for secreted proteins from fungi [51]. Some cellulases purified from strain JU1 had very similar amino acid compositions but different molecular weights, indicating that they might be encoded by the same gene but underwent diverse post­translational modifications [34]. On the 2D electrophoresis map of cultured supernatant, 106 secreted protein spots were assigned into a total of 38 protein models [20], which clearly confirmed the hypothesis of post­translation modification. The map also showed that most isoforms of one protein had similar molecular weights but differed greatly in isoelectric points.

The diversity of N­glycosylation on CBHI (Cel7A­2) from P. oxalicum was studied in detail [35]. Four glycoforms of CBHI with identical amino acid sequences but different N­glycan structures were purified from JU­A10. The four proteins had different specific activities, optimum temperatures and optimum pHs. In particular, one of the glycoforms, CBHI­A (carrying

Key terms

Product inhibition: Inhibition of the activity of an enzyme by its product.

Simultaneous saccharification and fermentation: Method for biomass conversion in which hydrolytic enzymes and fermenting microorganisms (e.g., yeast) are simultaneously added into the reactor for substrate utilization.

Glycoside hydrolase: Type of enzyme hydrolyzing glycosidic bonds. Glycoside hydrolases are classified into more than 100 families in the Carbohydrate-Active Enzyme database based on amino acid sequence similarities.

Cellulose binding domain: Amino acid sequence folding independently and showing cellulose-binding activity. Cellulose binding domains are usually found in cellulases and hemicellulases.

Post-translational modification: Processing of polypeptides after translation. The modifications include folding, cutting and adding chemical groups (e.g., phosphate and carbohydrates).

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[Man]3 [GlcNAc]

2 + GlcNAc at

Asn137), showed a signif icant synergism on cellulose degradation with commercial enzyme systems. CBHI­A had no hydrolytic activity on cellulose and p­nitrophenyl­b­d­cellobioside (a commonly used substrate for CBH assays), but could decrease the hydrogen bond intensity and crystalline degree of cotton fibers. It is reasonable to suppose that the properties of other enzyme components are also affected by diverse glycosylations.

The cellular machine for lignocellulolytic enzyme production in P. oxalicumSome sequenced filamentous fungi, such as Aspergillus niger, Aspergillus oryzae and Penicillium chrysogenum, have comparable or even higher numbers of lignocellulolytic enzyme­encoding genes than P. oxalicum [20]; however, they are not good producers of lignocellulolytic enzymes. This indicates that P. oxalicum holds more efficient transcription, translation or secretion machines for the production of lignocellulolytic enzymes.

Generally, lignocellulolytic enzymes in filamentous fungi are produced at high levels only in the presence of inducers (e.g., cellulose) [52]. The same feature was observed in P. oxalicum. The effects of different carbon sources on lignocellulolytic enzyme production, and the involved regulatory proteins or pathways (Figure 4), have been investigated in P. oxalicum.

� Effects of different carbon sourcesWhen P. oxalicum was grown in medium with glycerol or glucose as the sole carbon source, low activities of cellulase and xylanase were detected, suggesting that some enzyme components are expressed at basal levels [20,33,53]. When lignocellulosic substrates (e.g., cellulose, xylan, xylose and wheat bran) were used as carbon sources, significantly higher lignocellulolytic enzyme activities were detected. The repressing effect of glucose and inductive effect of cellulose on the expression of cellulases and xylanases were verified at gene­transcriptional levels [54]. Lactose, which is used for cellulase induction in T. reesei in industry [55], also induces the transcription of cellulase genes in P. oxalicum. However, the inductive effect on cellulase gene transcription is not sustained and no improved

cellulase production was detected. It is assumed that glucose produced by lactose hydrolysis repressed cellulase gene expression quickly [54]. Another strong inducer of cellulase in T. reesei, sophorose (b­1,2­glucobiose) [56], cannot induce the production of cellulases in P. oxalicum [57]. It is also noted that the wild­type strain 114–2 and mutant JU­A10 respond to specific sugars differently [54]. Lactose does not induce cellulase gene transcription and even cannot support cell growth in JU­A10. By contrast, l­sorbose induces cellulase gene transcription in JU­A10 but not in 114–2. The mechanisms for these differences are as yet unknown.

� How the cell senses celluloseThe inductive effect of insoluble cellulose on cellulase expression is generally believed to be mediated by cellodextrins (cellulose hydrolytic product) [58,59]. Supplementation of a small amount of exogenous cellodextrins (0.05%) to 2% cellulose medium did lead to improved cellulase production in P. oxalicum 114–2 [60]. However, endogenous cellodextrins are hardly detected when cellulose is used as the inducer. This might be due to the gradual release and rapid catabolism (by BGLs) of cellodextrins [58]. BGL­deficient mutants were constructed to study the role of cellodextrins in cellulose­induced cellulase expression in P. oxalicum [23].

M12

140-12JU-A10-TJU-A10-1

JU-A10 GS2-15

JU1

JN15

UV11

114 114-2Isolation

Mutagenesis

Mutagenesis

Mutagenesis

Mutagenesis

Adaption in SASL Genome shuffling

M12

∆pku70

Figure 2. Genealogy of Penicillium oxalicum strains. Strains shown in light gray have been lost over the years. SASL: Spent ammonium sulfite liquor.

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Key terms

Transcription factor: Protein activating or inhibiting the transcription of DNA to RNA through binding to specific DNA sequences.

Inverse metabolic engineering: Concept in metabolic engineering first identifying the genetic basis of a desired phenotype and then introducing the genotype into another host.

Submerged fermentation: Microorganisms are cultured in liquid medium in a large tank to make useful products. The culture is constantly mixed by stirring and supply of sterile air is needed.

Deletion of the major extracellular BGL gene bgl1 resulted in remarkably higher cellodextrins in the culture broth, but did not enhance the expression of cellulases. Deletion of the major intracellular BGL gene bgl2 accumulated cellobiose in the cytoplasm along with significantly increased expression of cellulases. In the bgl2­disrupting mutant, exogenous cellobiose alone can induce the transcription of cellulase genes. These results indicated that cellobiose released from cellulose is an inducer for cellulase gene expression in P. oxalicum after

being transported into the cell. This assumption was further verified by that in cellobiose transporter­deficient

mutant, cellulose­induced cellulase expression was dramatically reduced [Li J, Liu G, Li Z, Qin Y, Qu Y, Unpublished

Data].A subsequent question is how the cellobiose signal

is transduced after entering the cell. Sophorose and gentiobiose (b­1,6­glucobiose) are supposed to be important in cellobiose­induced cellulase expr­ession in T. reesei and Penicillium purpurogenum, respectively, because they strongly induce cellulase gene expression and can be produced from cellobiose through transglycosylation by intracellular enzymes [61,62]. In P. oxalicum mutants with both bgl1 and bgl2 deleted, cellobiose, but not sophorose or gentiobiose, induced cellulase expression [61,62]. The result clearly suggests different mechanisms for cellulase induction among fungal species. The protein­sensing and transducing cellobiose signal has not been identified in fungi so far.

CBHs

EGs

PMOs

PDE_05445 (Cel7A-1)

PDE_07945 (Cel7A-2)

PDE_07124 (Cel6A)

PDE_07929 (Cel6B)

PDE_00507 (Cel5A)

PDE_09226 (Cel5B)

PDE_09969 (Cel5C)

PDE_05193 (Cel5D)

PDE_06439 (Cel12A)

PDE_03711

PDE_09267

PDE_02886

PDE_09014

PDE_00698

PDE_01261

PDE_06768

PDE_07928 (Cel45A)

PDE_05633 (Cel61A)

S

S

S

S

C

C

C

S C

S C

S C

S C

S C

S C

Glyco_hydro_7

Glyco_hydro_7

Glyco_hydro_12

Glyco_hydro_12

Glyco_hydro_12

Glyco_hydro_45

Glyco_hydro_61

Glyco_hydro_61

Glyco_hydro_61

Glyco_hydro_61

Glyco_hydro_7

Glyco_hydro_6

Cellulase (GH5)

Cellulase (GH5)

Cellulase (GH5)

Cellulase (GH5)

Cellulase (GH5)

Catalytic domainCBM_1

Cellulase (GH5) CBM_X2

Figure 3. The 18 cellulolytic enzymes predicted in Penicillium oxalicum. Domain compositions of the enzymes are shown according to the results of Pfam ana lysis [45]. C: Enzymes characterized for enzymatic properties; CBH: Cellobiohydrolase; CBM_1: Family 1 carbohydrate binding module (formerly known as fungal cellulose-binding domain); EG: Endoglucanase; PMO: Polysaccharide mono-oxygenase; S: Enzymes detected in experimental secretomic analyses.

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Some classical signaling pathways (e.g., G protein signaling) were reported to affect cellulase expression in T. reesei, although they did not directly transduce the cellulose/cellodextrin signal [63,64]. One heterotrimeric G protein a subunit coupled to cyclic adenosine monophosphate signaling, PGA3, negatively regulates cellulase gene transcription in P. oxalicum through a carbon source­independent manner [Hu Y, Liu G, Li Z,

Qin Y, Qu Y, Unpublished Data]. The regulatory role only occurs in the early stage of cultivation but does not affect cellulase production levels, suggesting it is not a dominant regulator in cellulose signaling.

� Transcriptional regulationRegulation of lignocellulolytic enzyme production is mainly accomplished by controlling the transcriptional levels of genes encoding these enzymes [52]. As reported in other fungi, transcriptions of most cellulases and hemicellulases in P. oxalicum are simultaneously induced or repressed (i.e., co­regulated) by carbon sources [54]. Orthologs of several transcription factors previously known to be involved in this regulation were annotated in P. oxalicum [20], some of which were functionally studied. As expected, the CCR factor CreA [19] and deubiquitinating enzyme CreB that stabilizes CreA [65] negatively regulates cellulase production in P. oxalicum under both repressing and inducing conditions. The regulatory roles of transcription factors ACEI (negative regulator of cellulase expression [66]), ClrB (activator of cellulase and xylanase expression [67]) and XlnR (activator of xylanase and cellulase expression [68]) were also confirmed in P. oxalicum [Li Z,Qu Y, Unpublished Data].

To get a systematic understanding of the transcription factors regulating cellulase expression, a single­gene deletion strain set covering more than 400 transcription factors was constructed in P. oxalicum. The above­mentioned strain, Dpku70 (Figure 2) [24], was used as the parent strain to facilitate the efficiency of targeted gene deletion. Some transcription factors, whose functions were not reported previously, were shown to strongly affect cellulase production. It appears that we are still far away from a complete understanding of the mechanisms controlling lignocellulolytic enzyme expression in fungi.

� Lessons from the comparison of wild-type & mutant strainsThe cellulase hyper­producing mutants obtained through long­term random mutagenesis are good materials for the study of inverse metabolic engineering [69]. The genome sequences of mutant JU­A10­T (Figure 2) and wild­type 114–2 were compared. A large number of sequence variations (average 1.4 single­nucleotide variations per kilobase pair) were found between the two strains, making it difficult to identify the critical

variations accounting for the different production levels. The secretomes and transcriptomes of the two strains were then compared. Integrative ana lysis of these results showed significantly downregulated expression of amylases and proteases in addition to upregulated expression of cellulases and hemicellulases in JU­A10­T. The result led us to investigate the function of the well­known amylase transcription activator AmyR (dramatically downregulated in JU­A10­T) in cellulase expression [70]. Interestingly, gene deletion suggested that AmyR plays a negative role in cellulase expression, even when starch was absent in the medium [19]. In addition, a frame­shift mutation in CreA was proved to underlie the CCR­resistant expression of cellulases in JU­A10. Mutations in the gene­promoter region of cellobiohydrolase Cel7A­1 was proved to contribute to its exceptional over­presentation in the secretome of JU­A10­T [19].

Genome­wide transcription ana lysis also provided some other clues about the hyper­producing phenotype in JU­A10­T (Figure 4). First, more genes in JU­A10­T are expressed at low levels in comparison to those in 114–2 [19]. That is to say, the cell factory of JU­A10­T concentrates on fewer biological processes. Second, the expression levels of genes for the pentose phosphate pathway (providing NADPH and precursors for amino acids biosynthesis), lysine and cysteine synthesis, ribosome and protein folding are upregulated in JU­A10­T, all facilitating the high­level synthesis of lignocellulolytic enzymes. Third, amino acid degradation and most secondary metabolisms are reduced in JU­A10­T, which may increase the fluxes and energy used for enzyme synthesis. The results provide implications for future rational design of strains for higher production of lignocellulolytic enzymes.

Application of P. oxalicum enzyme systems in biorefinery

� Low-cost production of lignocellulolytic enzymesOne of the approaches for improving the economics of cellulosic biofuels production is to lower the cost of cellulase production. Submerged fermentation conditions were optimized for cellulase production by P. oxalicum. The optimal temperature and pH of cellulase production by strain JU1 is 28°C and 3–4, respectively [27]. Wheat bran, an agricultural residue mainly consisting of xylan, starch, crude protein and cellulose, has a remarkably stimulative effect on cellulase production by P. oxalicum [53,60]. Industrial wastes such as SASL, paper mill waste fiber, corncob residues (from xylo­oligosaccharide production) and acidic wastewater (from monosodium glutamate process) were used to substitute for part or all of the cultivation medium to

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further lower the cost of cellulase production [14,16,18]. Fed­batch fermentation was used to improve the volumetric cellulase activity in submerged fermentation [14]. Also, cellulase production by P. oxalicum in solid-state fermentation was studied, primarily focusing on the control of air flow [71–73].

As a combined result of strain improvement and process optimization, an extracellular protein concentration of 16.9 g l­1 and cellulase activity of 15.4

filter paper unit (FPU) ml­1 at 96 h of fermentation (i.e., productivity of 160 FPU l­1 h­1) was obtained for P. oxalicum mutant JU­A10–1. The mutant has been used for industrial production of cellulase preparations for feed, food and the textile industry in China since 1996 [74]. Also, the

P. oxalicum enzyme systems were used in research on pulping for high xylanase activity [75] and extraction of plant flavonoids for high transglycosylation activity [76].

� Biorefinery of agricultural residues using P. oxalicum enzymesWe have developed a technology route for the biorefinery of nonwood lignocelluloses with pulp, ethanol and single­cell protein as the main products [77]. P. oxalicum was used for cellulase production in this process using wastes from pulping as the culture medium. Biorefinery of corncob was proposed based on the previously well­established production of xylose, xylitol, xylo­oligosaccharides and furfural, with ethanol and lignin as the co­products [77]. In this process, corncob residues (containing mainly cellulose and lignin [17]) and wheat bran were used as the main carbon sources for the on­site

Lignocellulose

Cellulases Hemicellulases

Amylases, and so on

BGLs

Cellodextrins Glucose Pentoses

BGLsCellodextrins Glucose

Glycolysis

PPP

NADPH

Amino acidsynthesis

Citrate cycle

Secondary metabolism

Amino acidcatabolism

Amino acids

Protein synthesis

Protein folding

ClrB, XlnR, and so on

AmyR CreA

Nucleus Cytoplasm

(hemi-)cel

amy

others

GTsCDTs

?

PTs

Figure 4. Summary of proteins and cellular processes involved in cellulase production in Penicillium oxalicum. Black arrows indicate mass flows and gray arrows indicate regulatory interactions. Genes, proteins or pathways of remarkable expression changes in JU-A10-T compared with those in 114–2 are marked (triangle for upregulation and inverted triangle for downregulation). BGL: b-glucosidase; CDT: Cellodextrin transporter; GT: Glucose transporter; PPP: Pentose phosphate pathway; PT: Pentose transporter.

Key term

Solid-state fermentation: Microorganisms are cultured on solid medium (with reduced water activity) to make products. Solid-state fermentation has the advantage of producing some chemicals and enzymes.

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production of lignocellulolytic enzymes by P. oxalicum. The crude enzymes can be directly used for simultaneous saccharification and fermentation of corncob residues to ethanol without removing fungal cells. Thus, the cost of enzymes is further reduced. Using a fed­batch strategy, the concentration of ethanol produced from delignined corncob residues reached 57.2 g L­1 at a low­enzyme dosage (9.3 FPU g­1 substrate) after 141.5 h of fermentation [17]. At present, a corncob­based cellulosic ethanol project in a scale of 50,000 tonne per year has been built at Yucheng, China [101].

The negative role of lignin in the hydrolysis of complex lignocellulosic materials by P. oxalicum enzymes was found [17]. When delignined corncob residues were used as the substrate, significantly higher concentration of ethanol was produced in comparison to that using corncob residues as the substrate. Electrophoresis ana lysis indicated that the major cellulase CBHI was irreversibly adsorbed to the lignin, which might reduce its efficiency in cellulose hydrolysis. CBHIs from Penicillium pulvillorum and T. reesei were recently shown to be differently adsorbed to lignin [7], indicating the possibility to reduce the adsorption of CBHI to lignin by protein engineering in the future.

Future perspectiveAlthough many strategies have been employed, the performance and production level of lignocellulolytic enzyme systems produced by P. oxalicum still need

to be further improved to benefit the economy. For example, supplementation of exogenous BGL could further improve the performance of P. oxalicum enzyme systems in cellulosic ethanol production [78], despite its relatively higher BGL activity (compared with that of T. reesei). Some other components (e.g., CBHI, which binds to lignin) in the enzyme system also need to be optimized for the hydrolysis of different lignocellulolytic materials. On the other hand, the enzyme production level in P. oxalicum is still lower than those reported in T. reesei and some other fungi (up to 100 g l­1 [49,79]). Based on the results from systems biology studies, the production level in P. oxalicum is expected to be further elevated. To achieve this goal, understanding of the related cellular processes (e.g., signal transduction, transcription regulation and protein secretion) is being enhanced and highly efficient genetic manipulation tools (e.g., multiple­gene targeting) are being developed.

Financial & competing interests disclosureThis work was supported by grants from the National Basic Research Program of China (grant no. 2011CB707403) and the National Natural Sciences Foundation of China (grant no. 31030001). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Executive summary

Screening and development of Penicillium oxalicum strains for cellulase production � The wild-type P. oxalicum isolate 114 was screened from decayed straw-covered soil using double-layer agar plates containing ball-milled

holocellulose. � A series of carbon catabolite repression-resistant mutants with higher cellulase production levels were obtained through random

mutagenesis. � Genetic engineering of P. oxalicum was performed based on genomic ana lysis.

Characterization of the lignocellulolytic enzyme system of P. oxalicum � Some enzyme components were studied prior to genome sequencing through protein purification and gene cloning. � Integrated genomic and proteomic ana lysis showed that the enzyme system of P. oxalicum contained more diverse components than that

of Trichoderma reesei. � Post-translational modification widely occurs on lignocellulolytic enzymes and affects their activities.

The cellular machine for lignocellulolytic enzyme production in P. oxalicum � Glucose represses, while cellulose induces, the expression of lignocellulolytic enzymes. Lactose and sophorose cannot induce high-level

cellulase production in P. oxalicum. � Intracellular cellodextrins are important in the induction of lignocellulolytic enzyme expression by cellulose. � In addition to several well-documented regulators, novel transcription factors are found to be involved in the transcriptional regulation of

lignocellulolytic enzymes. � Comparative ana lysis of wild-type and mutant strains identified critical factors (including transcription factors CreA and AmyR) affecting

cellulase production level.Application of P. oxalicum enzyme systems in biorefinery

� Fermentation conditions were optimized and low-cost media were designed for the production of lignocellulolytic enzymes. � P. oxalicum enzymes were used in the biorefinery of corncob in China.

Future perspective � The hydrolysis performance and production level of P. oxalicum enzyme systems still need to be improved in the future.

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ReferencesPapers of special note have been highlighted as:n of interestnn of considerable interest

1 Merino ST, Cherry J. Progress and challenges in enzyme development for biomass utilization. Adv. Biochem. Eng. Biotech. 108, 95–120 (2007).

2 Liu G, Qin Y, Li Z, Qu Y. Development of highly efficient, low­cost lignocellulolytic enzyme systems in the post­genomic era. Biotechnol. Adv. doi:10.1016/j.biotechadv.2013.03.001 (2013) (Epub ahead of print).

3 Klein­Marcuschamer D, Oleskowicz­Popiel P, Simmons BA, Blanch HW. The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol. Bioeng. 109(4), 1083–1087 (2011).

4 Seidl V, Seiboth B. Trichoderma reesei: genetic approaches to improving strain efficiency. Biofuels 1(2), 343–354 (2010).

n Review of the history, current knowledge and available genetic tools in T. reesei.

5 Kubicek CP, Mikus M, Schuster A, Schmoll M, Seiboth B. Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina. Biotechnol. Biofuels 2, 19 (2009).

n Mechanisms for carbon sensing, signal transduction and transcriptional regulation in cellulase production by Trichoderma reesei.

6 Gusakov AV, Sinitsyn AP. Cellulases from Penicillium species for producing fuels from biomass. Biofuels 3(4), 463–477 (2012).

nn Comprehensive review on cellulase production by Penicillium species.

7 Marjamaa K, Toth K, Bromann PA, Szakacs G, Kruus K. Novel Penicillium cellulases for total hydrolysis of lignocellulosics. Enzyme Microb. Tech. 52(6–7), 358–369 (2013).

8 Chekushina A, Dotsenko G, Sinitsyn A. Comparing the efficiency of plant material bioconversion processes using biocatalysts based on Trichoderma and Penicillium verruculosum enzyme preparations. Catal. Ind. 5(1), 98–104 (2013).

9 Sahare P, Singh R, Laxman RS, Rao M. Effect of alkali pretreatment on the structural properties and enzymatic hydrolysis of corn cob. Appl. Biochem. Biotech. 168(7), 1806–1819 (2012).

10 Guais O, Borderies G, Pichereaux C et al. Proteomics analysis of ‘Rovabiot Excel’, a secreted protein cocktail from the filamentous

fungus Penicillium funiculosum grown under industrial process fermentation. J. Ind. Microbiol. Biotech. 35(12), 1659–1668 (2008).

11 Larkin MA, Blackshields G, Brown NP et al. Clustal W and Clustal X version 2.0. Bioinformatics 23(21), 2947–2948 (2007).

12 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28(10), 2731–2739 (2011).

13 Qu Y, Gao P, Wang Z. Screening of catabolite repression­resistant mutants of cellulase producing Penicillium spp. Acta. Mycol. Sin. 3, 238–243 (1984).

14 Qu Y, Zhao X, Gao P, Wang Z. Cellulase production from spent sulfite liquor and paper­mill waste fiber. Appl. Biochem. Biotech. 28–29, 363–368 (1991).

15 Zhao X, Qu Y, Gao P. The discussion on the selection of a cellulase­production strain in black liquor. J. Cellulose Sci. Technol. 1(2), 28–32 (1993).

16 Cheng Y, Song X, Qin Y, Qu Y. Genome shuffling improves production of cellulase by Penicillium decumbens JU­A10. J. Appl. Microbiol. 107(6), 1837–1846 (2009).

17 Liu K, Lin X, Yue J et al. High concentration ethanol production from corncob residues by fed­batch strategy. Bioresource Technol. 101(13), 4952–4958 (2010).

18 Yao L, Yue J, Zhao J, Dong J, Li X, Qu Y. Application of acidic wastewater from monosodium glutamate process in pretreatment and cellulase production for bioconversion of corn stover – feasibility evaluation. Bioresource Technol. 101(22), 8755–8761 (2010).

19 Liu G, Zhang L, Qin Y et al. Long­term strain improvements accumulate mutations in regulatory elements responsible for hyper­production of cellulolytic enzymes. Sci. Rep. 3, 1569 (2013).

n Describes alternations in the lignocellulolytic enzyme system and related regularory factors found by comparing Penicillium oxalicum mutant with a wild-type strain.

20 Liu G, Zhang L, Wei X et al. Genomic and secretomic analyses reveal unique features of the lignocellulolytic enzyme system of Penicillium decumbens. PLoS ONE 8(2), e55185 (2013).

nn Genome-wide annotation and experimental analysis of the P. oxalicum enzyme system.

21 Tian C, Beeson WT, Iavarone AT et al. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proc. Natl Acad. Sci. USA 106(52), 22157–22162 (2009).

22 Zhang Y, Zhang J, Xiao P, Wang T, Qu Y. Improved cellulase production via disruption of PDE01641 in cellulolytic fungus Penicillium decumbens. Bioresource Technol. 123, 733–737 (2012).

23 Chen M, Qin Y, Cao Q et al. Promotion of extracellular lignocellulolytic enzymes production by restraining the intracellular b­glucosidase in Penicillium decumbens. Bioresource Technol. 137, 33–40 (2013).

24 Li Z, Du C, Zhong Y, Wang T. Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens. Appl. Microbiol. Biotech. 87(3), 1065–1076 (2010).

25 Qin Y, Zheng K, Liu G, Chen M, Qu Y. Improved cellulolytic efficacy in Penicilium decumbens via heterologous expression of Hypocrea jecorina endoglucanase II. Arch. Biol. Sci. Belgrade 65(1), 305–314 (2013).

26 Steiger MG, Vitikainen M, Uskonen P et al. Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers. Appl. Environ. Microbiol. 77(1), 114–121 (2011).

27 Qu Y, Gao P, Wang Z. Studies on the cellulase system of Penicillium decumbens I. The conditions of enzyme production and reaction of mutant JU1. J. Shandong Univ. 21(1), 140–143 (1986).

28 De Castro AM, De Albuquerque De Carvalho ML, Leite SG, Pereira N Jr. Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis. J. Ind. Microbiol. Biotech. 37(2), 151–158 (2010).

29 Castellanos O, Sinitsyn A, Vlasenko EY. Comparative evaluation of hydrolytic efficiency toward microcrystalline cellulose of Penicillium and Trichoderma cellulases. Bioresource Technol. 52(2), 119–124 (1995).

30 Busto M, Ortega N, Perez­Mateos M. Location, kinetics and stability of cellulases induced in Trichoderma reesei cultures. Bioresource Technol. 57(2), 187–192 (1996).

31 Wang M, Mu Z, Wang J et al. The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in

Improving lignocellulolytic enzyme production with Penicillium Review

future science group www.future-science.com 533

cellulose saccharification catalyzed by cellulases from Penicillium decumbens. Bioresource Technol. 133, 507–512 (2013).

32 Zhang YH, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol. Bioeng. 88(7), 797–824 (2004).

33 Sun X, Liu Z, Zheng K, Song X, Qu Y. The composition of basal and induced cellulase systems in Penicillium decumbens under induction or repression conditions. Enzyme Microb. Technol. 42(7), 560–567 (2008).

34 Qu Y, Gao P, Wang Z. Studies on cellulase system from Penicillium decumbens. Acta Microbiol. Sin. 28(2), 121–130 (1988).

35 Gao L, Gao F, Wang L et al. N­glycoform diversity of cellobiohydrolase I from Penicillium decumbens and the synergism of a nonhydrolytic glycoform in cellulose degradation. J. Biol. Chem. 287(19), 15906–15915 (2012).

36 Gao L, Wang F, Gao F, Wang L, Zhao J, Qu Y. Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU­A10 for bioethanol production. Bioresource Technol. 102(17), 8339–8342 (2011).

37 Liu Z, Sun X, Qu Y. Cloning cellobiohydrolase I from Penicillium decumbens 114–2 with TAIL­PCR and comparing with its derepressed mutant JU­A10. Acta. Microbiol. Sin. 48(5), 667–671 (2008).

38 Liu G, Wei X, Qin Y, Qu Y. Characterization of the endoglucanase and glucomannanase activities of a glycoside hydrolase family 45 protein from Penicillium decumbens 114–2. J. Gen. Appl. Microbiol. 56(3), 223–229 (2010).

39 Wei X, Qin Y, Qu Y. Molecular cloning and characterization of two major endoglucanases from Penicillium decumbens. J. Microbiol. Biotechnol. 20(2), 265–270 (2010).

40 Gruno M, Valjamae P, Pettersson G, Johansson G. Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate. Biotechnol. Bioeng. 86(5), 503–511 (2004).

41 Shen Y, Zhang Y, Ma T et al. Simultaneous saccharification and fermentation of acid­pretreated corncobs with a recombinant Saccharomyces cerevisiae expressing b­glucosidase. Bioresource Technol. 99(11), 5099–5103 (2008).

42 Chen M, Qin Y, Liu Z, Liu K, Wang F, Qu Y. Isolation and characterization of a b­glucosidase from Penicillium decumbens and improving hydrolysis of corncob residue by

using it as cellulase supplementation. Enzyme Microb. Technol. 46(6), 444–449 (2010).

43 Ma L, Zhang J, Zou G, Wang C, Zhou Z. Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a b­glucosidase gene from Penicillium decumbens. Enzyme Microb. Technol. 49(4), 366–371 (2011).

44 Phillips CM, Beeson WT, Cate JH, Marletta MA. Cellobiose dehydrogenase and a copper­dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem. Biol. 6(12), 1399–1406 (2011).

45 Punta M, Coggill PC, Eberhardt RY et al. The Pfam protein families database. Nucleic Acids Res. 40, D290–D301 (2012).

46 Liu G, Qin Y, Hu Y, Gao M, Peng S, Qu Y. An endo­1,4­b­glucanase PdCel5C from cellulolytic fungus Penicillium decumbens with distinctive domain composition and hydrolysis product profile. Enzyme Microb. Technol. 52(3), 190–195 (2013).

47 Herpoel­Gimbert I, Margeot A, Dolla A et al. Comparative secretome analyses of two Trichoderma reesei RUT­C30 and CL847 hypersecretory strains. Biotechnol. Biofuels 1(1), 18 (2008).

48 Chundawat SP, Lipton MS, Purvine SO et al. Proteomics­based compositional analysis of complex cellulase–hemicellulase mixtures. J. Proteome Res. 10(10), 4365–4372 (2011).

49 Martinez D, Berka RM, Henrissat B et al. Genome sequencing and analysis of the biomass­degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26(5), 553–560 (2008).

50 Krogh K, Kastberg H, Jørgensen C, Berlin A, Harris P, Olsson L. Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins. Enzyme Microb. Technol. 44(6), 359–367 (2009).

51 Bouws H, Wattenberg A, Zorn H. Fungal secretomes – nature’s toolbox for white biotechnology. Appl. Microbiol. Biotechnol. 80(3), 381–388 (2008).

52 Aro N, Pakula T, Penttila M. Transcriptional regulation of plant cell wall degradation by filamentous fungi. FEMS Microbiol. Rev. 29(4), 719–739 (2005).

53 Qu Y, Gao P, Wang Z. Studies on the cellulase system of Penicillium decumbens II. Physiological characters of the mutant JU1 and regulation of its enzymes synthesis. J. Shandong Univ. 22(13), 97–104 (1987).

54 Wei X, Zheng K, Chen M et al. Transcription analysis of lignocellulolytic enzymes of Penicillium decumbens 114–2 and its

catabolite­repression­resistant mutant. C. R. Biol. 334(11), 806–811 (2011).

55 Warzywoda M, Ferre V, Pourquie J. Development of a culture medium for large­scale production of cellulolytic enzymes by Trichoderma reesei. Biotechnol. Bioeng. 25(12), 3005–3011 (1983).

56 Sternberg D, Mandels GR. Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J. Bacteriol. 139(3), 761–769 (1979).

57 Wang D, Qu Y, Gao P. Regulation of cellulase synthesis in mycelial fungi: participation of ATP and cyclic AMP. Biotechnol. Lett. 17(6), 593–598 (1995).

58 Znameroski EA, Coradetti ST, Roche CM et al. Induction of lignocellulose­degrading enzymes in Neurospora crassa by cellodextrins. Proc. Natl Acad. Sci. USA 109(16), 6012–6017 (2012).

59 Schmoll M, Kubicek CP. Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol. Immunol. Hung. 50(2–3), 125–145 (2003).

60 Sun X, Liu Z, Qu Y, Li X. The effects of wheat bran composition on the production of biomass­hydrolyzing enzymes by Penicillium decumbens. Appl. Biochem. Biotechnol. 146(1–3), 119–128 (2008).

61 Vaheri M, Leisola M, Kauppinen V. Transglycosylation products of cellulase system of Trichoderma reesei. Biotechnol. Lett. 1(1), 41–46 (1979).

62 Kurasawa T, Yachi M, Suto M, Kamagata Y, Takao S, Tomita F. Induction of cellulase by gentiobiose and its sulfur­containing analog in Penicillium purpurogenum. Appl. Environ. Microbiol. 58(1), 106–110 (1992).

63 Schmoll M, Schuster A, Silva Rdo N, Kubicek CP. The G­alpha protein GNA3 of Hypocrea jecorina (anamorph Trichoderma reesei) regulates cellulase gene expression in the presence of light. Eukaryot. Cell 8(3), 410–420 (2009).

64 Zhang J, Zhang Y, Zhong Y, Qu Y, Wang T. Ras GTPases modulate morphogenesis, sporulation and cellulase gene expression in the cellulolytic fungus Trichoderma reesei. PLoS ONE 7(11), e48786 (2012).

65 Zhou G, Lv J, Li Z et al. Enhanced cellulase production of Penicillium decumbens by knocking out creB encoding a deubiquitination enzyme. Chin. J. Biotech. 28(8), 959–972 (2012).

66 Aro N, Ilmen M, Saloheimo A, Penttila M. ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression. Appl. Environ. Microbiol. 69(1), 56–65 (2003).

Biofuels (2013) 4(5) future science group534

Review Liu, Qin, Li & Qu

67 Coradetti ST, Craig JP, Xiong Y, Shock T, Tian C, Glass NL. Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi. Proc. Natl Acad. Sci. USA 109(19), 7397–7402 (2012).

n Describes the discovery of crucial transcription factors regulating cellulase expression by screening the single-gene deletion mutant set of model fungus Neurospora crassa.

68 Van Peij NN, Visser J, De Graaff LH. Isolation and analysis of xlnR, encoding a transcriptional activator co­ordinating xylanolytic expression in Aspergillus niger. Mol. Microbiol. 27(1), 131–142 (1998).

69 Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA, Tsai PS. Inverse metabolic engineering: a strategy for directed genetic engineering of useful phenotypes. Biotechnol. Bioeng. 52(1), 109–121 (1996).

70 Petersen KL, Lehmbeck J, Christensen T. A new transcriptional activator for amylase genes in Aspergillus. Mol. Gen. Genet. 262(4–5), 668–676 (1999).

71 Mo H, Zhang X, Li Z. Control of gas phase for enhanced cellulase production by Penicillium decumbens in solid­state culture. Process Biochem. 39(10), 1293–1297 (2004).

72 Xu F, Chen H, Li Z. Effect of periodically dynamic changes of air on cellulase production in solid­state fermentation. Enzyme Microb. Technol. 30(1), 45–48 (2002).

73 Zhang X, Mo H, Zhang J, Li Z. A solid­state bioreactor coupled with forced aeration and pressure oscillation. Biotechnol. Lett. 25(5), 417–420 (2003).

74 Fang X, Shen Y, Zhao J, Bao X, Qu Y. Status and prospect of lignocellulosic bioethanol production in China. Bioresource Technol. 101(13), 4814–4819 (2010).

75 Zhao J, Li X, Qu Y. Application of enzymes in producing bleached pulp from wheat straw. Bioresource Technol. 97(13), 1470–1476 (2006).

76 Chen S, Xing X, Huang J, Xu M. Enzyme­assisted extraction of flavonoids from Ginkgo biloba leaves: improvement effect of flavonol

transglycosylation catalyzed by Penicillium decumbens cellulase. Enzyme Microb. Technol. 48(1), 100–105 (2011).

77 Qu Y, Zhu M, Liu K, Bao X, Lin J. Studies on cellulosic ethanol production for sustainable supply of liquid fuel in China. Biotechnol. J. 1(11), 1235–1240 (2006).

78 Zhao X, Qu Y, Gao P. Acceleration of ethanol production from paper mill waste fiber by supplementation with b­glucosidase. Enzyme Microb. Technol. 15(1), 62–65 (1993).

79 Visser H, Joosten V, Punt PJ et al. Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1. Ind. Biotechnol. 7(3), 214–223 (2011).

� Website101 Green Prospects Asia. Shandong longlive

bio­technology delivers first batch of fuel ethanol from corncobs. www.greenprospectsasia.com/content/shandong­longlive­bio­technology­delivers­first­batch­fuel­ethanol­corncobs